Polarizing Region Grafts Fig 3C see Note

1. Remove chick embryo from egg, and place in medium (as in Subheading 3.1.).

2. Treat chick limb buds (stages 19-21) with cold trypsin and remove ectoderm.

3. Polarizing region in chick wing buds has been mapped from stages 17-29, and with reference to these maps of polarizing activity (9-11), cut appropriate region out of mesenchymal core of limb bud with sharp needles. A piece of tissue about 200 x 200 |im2 from the thickness of the limb bud is easy to handle, although smaller pieces can be used.

4. Prepare host embryo by peeling away vitelline membranes and amnion over right wing bud. (Host embryos can be used between stages 18 and 24 but graft must be placed in contact with the ridge; to obtain complete digit duplications do not use stages later than 20/21; ref. 32).

5. Make site for graft by cutting along base of apical ridge over anterior margin of right wing bud with sharpened needle. First make a series a short superficial cuts through the dorsal ectoderm and into the underlying mesenchyme. The cuts are made just below the junction between the transparent ridge and opaque mesenchyme working from the bud apex toward the anterior edge of the bud (Fig. 3C). Then push needle through to the ventral side of wing bud, and repeat process working along slit. Pull loosened apical ridge away from mesenchyme to make a loop. The apical ridge can be stretched, but it will gradually shrink back against the mesenchyme.

6. Transfer polarizing tissue graft into host egg using either a glass or gilson pipet or a very small spatula.

7. Maneuver graft under apical ridge loop using the end of a needle. The apical ridge will hold graft in place.

8. Seal egg with sellotape.

9. It is often useful to cut a small hole in the sellotape window the next day, so that graft and limb bud can be inspected. It is usually possible to see whether the graft has remained in place.

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